1. Field of the Invention
This invention relates to novel theophylline derivatives pertaining to immunoassays for determining theophylline in liquid media such as biological fluids. Such derivatives include theophylline immunogens used to stimulate production of antibodies to theophylline in host animals by conventional techniques. Also provided are labeled theophylline conjugates used as reagents, along with the theophylline antibodies, in particularly preferred immunoassays. Intermediates in the synthesis of the aforementioned immunogens and labeled conjugates are also provided.
Theophylline [1,3-dimethylxanthine, cf. The Merck Index, 9th ed., p. 1196(1976)] is a drug of great value in the management of asthmatic patients. The drug is used prophylactically, as an adjunct in the treatment of prolonged asthmatic attacks and in the management of status asthmaticus [Goodman and Gilman, The Pharmacological Basis of Therapeutics, 5th ed., MacMillan (New York 1975) p. 375]. However, because of its potential toxicity, proper dosage administration is critical. Frequent testing of serum or plasma concentrations assists in maintaining therapeutic levels and avoiding toxic levels.
2. Description of the Prior Art
The determination of theophylline by immunoassay is well known. Commercial test kits are available for measuring serum and plasma theophylline levels by radioimmunoassay, enzyme immunoassay, and substrate-labeled fluorescent immunoassay. Radioimmunoassays for determining theophylline are described by Cook et al, Res. Commun. Chem. Path. Pharm. 13:497(1976), Nishikawa et al, Chem. Pharm. Bull. 27:893(1979), and German OLS 2,901,281. Reagents for use in an enzyme immunoassay for determining theophylline are described in U.S. Pat. No. 4,230,805. The determination of theophylline by substrate-labeled fluorescent immunoassay (SLFIA) is described in U.S. Pat. No. 4,279,992. See also British Pat. No. 1,552,607 and Li et al, Clin. Chem. 27:22(1981).
Theophylline immunogens have been prepared by coupling the drug at various positions on its ring structure to conventional immunogenic carrier materials. Immunogens prepared by derivatization of theophylline at its 8-position are described by Cook et al and Li et al, supra, and in U.S. Pat. No. 4,279,992. Derivatization at the 7-position is known from Nishakawa et al supra, and German OLS No. 2,901,218. U.S. Pat. No. 4,230,805 describes the coupling of theophylline at its 3-position.
The state-of-the-art of preparing antibodies to haptens such as drugs is represented by Weinryb et al, Drug Metabolism Reviews 10:271(1979); Playfair et al, Br. Med. Bull. 30:24(1974); Broughton et al, Clin. Chem. 22:726(1976); and Butler, J. Immunol. Meth. 7:1(1975). The preparation of 9-xanthine derivatives are described by Lister, Purines, Wiley, Interscience (New York 1971) pp. 223-228; Hurst, An Introduction to the Chemistry and Biochemistry of the Pyrimidines, Purines, and Pteridines, John Wiley & Sons (New York 1980) p. 66; Rybar et al, Collect. Czech. Chem. Commun. 37:3936(1972); Fleiderer and Nubel, Justus Liebig's Annalen 631:168(1960); von Schuk, German Pat. No. 1,113,696 (Chem. Abst. 56P:12910e); and Blicke and Schaff, J. Amer. Chem. Soc. 78:5857(1956).